Inhibitors of nucleoside metabolism

ABSTRACT

The present invention provides compounds having the formula: ##STR1## wherein A is CH or N; B is chosen from OH, NH 2 , NHR, H or halogen; D is chosen from OH, NH 2 , NHR, H, halogen or SCH 3  ; R is an optionally substituted alkyl, aralkyl or aryl group; and X and Y are independently selected from H, OH or halogen except that when one of X and Y is hydroxy or halogen, the other is hydrogen; and Z is OH or, when X is hydroxy, Z is selected from hydrogen, halogen, hydroxy, SQ or OQ, Q is an optionally substituted alkyl, aralkyl or aryl group; or a tautomer thereof; or a pharmaceutically acceptable salt thereof; or an ester thereof; or a prodrug thereof. The present invention also provides the use of these compounds as pharmaceuticals, pharmaceutical compositions containing the compounds and processes for preparing the compounds.

TECHNICAL FIELD

The invention relates to certain nucleoside analogues, the use of thesecompounds as pharmaceuticals, pharmaceutical compositions containing thecompounds and processes for preparing the compounds.

BACKGROUND ART

Purine nucleoside phosphorylase (PNP) catalyses the phosphorolyticcleavage of the ribo- and deoxyribonucleosides of guanine andhypoxanthine to give the corresponding sugar-1-phosphate and guanine orhypoxanthine.

Humans deficient in purine nucleoside phosphorylase (PNP) suffer aspecific T-cell immunodeficiency due to an accumulation of dGTP and itstoxicity to stimulated T lymphocytes. Because of this, inhibitorsagainst PNP are immunosuppressive, and are active against T-cellmalignancies. Clinical trials are now in progress using9-(3-pyridylmethyl)-9-deazaguanine in topical form against psoriasis andin oral form for T-cell lymphoma and immunosuppression (BioCrystPharmaceuticals, Inc). The compound has an IC₅₀ of 35 nM for the enzyme.In animal studies, a 50 mg/kg oral dose is required for activity in acontact sensitivity ear swelling assay in mice. For human doses, thiswould mean approximately 3.5 grams for a 70 kg human. With thisinhibitor, PNP is difficult to Inhibit due to the relatively highactivity of the enzyme in blood and mammalian tissues.

Nucleoside hydrolases catalyse the hydrolysis of nucleosides. Theseenzymes are not found in mammals but are required for nucleoside salvagein some protozoan parasites. Some protozoan parasites use nucleosidephosphorylases instead or as well for this purpose. Inhibitors ofnucleoside hydrolases and phosphorylases can be expected to interferewith the metabolism of the parasite and therefore be usefully employedagainst protozoan parasites.

It is an object of the invention to provide pharmaceuticals which arevery effective inhibitors of PNP and/or nucleoside hydrolases.

DISCLOSURE OF THE INVENTION

In one aspect the invention provides compounds having the formula:##STR2## wherein A is CH or N; B is chosen from OH, NH₂, NHR, H orhalogen; D is chosen from OH, NH₂, NHR, H, halogen or SCH₃ ; R is anoptionally substituted alkyl, aralkyl or aryl group; and X and Y areindependently selected from H, OH or halogen except that when one of Xand Y is hydroxy or halogen, the other Is hydrogen; and Z is OH or, whenX is hydroxy, Z is selected from hydrogen, halogen, hydroxy, SQ or OQ, Qis an optionally substituted alkyl, aralkyl or aryl group; or a tautomerthereof; or a pharmaceutically acceptable salt thereof; or an esterthereof; or a prodrug thereof.

Preferably when either of B and/or D is NHR, then R is C₁ -C₄ alkyl.

Preferably when one or more halogens are present they are chosen fromchlorine and fluorine.

Preferably when Z is SQ or OQ, Q is C₁ -C₅ alkyl or phenyl.

Preferably D is H, or when D is other than H, B is OH.

More preferably, B is OH, D is H, OH or NH₂, X is OH or H, Y is H, mostpreferably with Z as OH, H or methylthio, especially OH.

It will be appreciated that the representation of a compound of formula(I) wherein B and/or D is a hydroxy group used herein is of theenol-type tautomeric form of a corresponding amide, and this willlargely exist in the amide form. The use of the enol-type tautomericrepresentation is simply to allow fewer structural formulae to representthe compounds of the invention.

Particularly preferred are the following compounds:

1. (1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol

2.(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitol

3.(1R)-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol

4.(1S)-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol

5.(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-5-methylthio-D-ribitol

6.(1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol

7.(1R)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol

8.(1S)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol

9.(1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-5-methylthio-D-ribitol

10.(1R)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol

11.(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol

12.(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol

13.(1S)-1,4-dideoxy-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-D-ribitol

14.(1R)-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol

15.(1S)-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol

16.(1S)-1,4-dideoxy-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-5-methylthio-D-ribitol

17.(1S)-1,4-dideoxy-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-D-ribitol

18.(1R)-1-C-5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol

19.(1S)-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol

20.(1S)-1,4-dideoxy-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-5-methylthio-D-ribitol

21.(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-D-ribitol

22.(1R)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol

23.(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol

24.(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol

Most preferred are compounds Ia and Ib, their tautomers andpharmaceutically acceptable salts. ##STR3##

According to another aspect of the invention, there is provided apharmaceutical composition comprising a pharmaceutically effectiveamount of a compound of the first aspect of the invention.

Preferably the pharmaceutical composition comprises a compound chosenfrom the preferred compounds of the first aspect of the invention; morepreferably the compound is chosen from the more preferred compounds ofthe first aspect. Most preferably the compound is the compound offormula Ia or Ib.

In another aspect the invention provides methods for treatment ofdiseases or conditions in which it is desirable to decrease the level ofT lymphocyte activity. The methods comprise administering apharmaceutically effective dose of a compound of the invention to apatient requiring treatment.

The diseases include T-cell malignancies and autoimmune diseasesincluding arthritis and lupus. This aspect of the invention alsoincludes use of the compounds for immunosuppression for organtransplantation and for inflammatory disorders. The invention includesuse of the compounds for manufacture of medicaments for thesetreatments.

In another aspect the invention provides a method for treatment and/orprophylaxis of parasitic infections, particularly those caused byprotozoan parasites. Included among the protozoan parasites are those ofthe genera Giardia, Trichomonas, Leishmania, Trypanosoma, Crithidia,Herpetomonas, Leptomonas, Histomonas, Eimeria, Isopora and Plasmodium.The method can be advantageously applied with any parasite containingone or more nucleoside hydrolases inhibited by the compound of theinvention when administered in a amount providing an effectiveconcentration of the compound at the location of the enzyme.

In another aspect the invention provides a method of preparing thecompounds of the first aspect of the invention. The method may includeone or more of methods (A)-(V).

Method (A):

(4-hydroxypyrrolo[3,2-d]pyrimidines and access to 5'-deoxy-,5'-deoxy-5'-halogeno-, 5'-ether and 5'-thio-analogues)

reacting a compound of formula (II) ##STR4## [wherein Z' is a hydrogenor halogen atom, a group of formula SQ or OQ, or a trialkylsilyloxy,alkyldiarylsilyloxy or optionally substituted triarylmethoxy group and Qis an optionally substituted alkyl, aralkyl or aryl group,]

(typically Z' is a tert-butyldimethylsilyloxy, trityloxy or similargroup)

sequentially with N-chlorosuccinimide then a sterically hindered base(such as lithium tetramethylpiperadide) to form an imine, then with theanion of acetonitrile (typically made by treatment of acetonitrile withn-butyllithium) followed by di-tert-butyl dicarbonate. This generates acompound of formula (III) ##STR5## [wherein Z' is as defined for formula(II) where first shown above]which is then elaborated following theapproach used to prepare 9-deazainosine [Lim et al., J. Org. Chem., 48(1983) 780] in which a compound of formula (III) is condensed with (Me₂N)₂ CHOBu^(t) and hydrolyzed under weakly acidic conditions to acompound of formula (IV) ##STR6## [wherein Z' is as defined for formula(II) where first shown above]which is then sequentially condensed with asimple ester of glycine (e.g. ethyl glycinate) under mildly basicconditions, cyclized by reaction with a simple ester of chloroformicacid (e.g. benzyl chloroformate or methyl chloroformate) and thendeprotected on the pyrrole nitrogen by hydrogenolysis in the presence ofa noble metal catalyst (e.g. Pd/C) in the case of a benzyl group orunder mildly basic conditions in the case of a simple alkyl group suchas a methyl group, to give a compound of formula (V) ##STR7## [whereinZ' is as defined for formula (II) where first shown above, and R is analkyl group]

(typically R is a methyl or ethyl group)

which is then condensed with formamidine acetate to give a compound offormula (VI) ##STR8## [wherein Z' is as defined for formula (II) wherefirst shown above]which is then fully deprotected under acidicconditions, e.g. by treatment with trifluoroacetic acid.

Methods for the preparation of a compound of formula (II) wherein Z' isa tert-butyldimethylsilyloxy group are detailed in Furneaux et al,Tetrahedron 53 (1997) 2915 and references therein.

A compound of formula (II) [wherein Z' is a halogen atom], can beprepared from a compound of formula (II) [wherein Z' is a hydroxygroup], by selective N-alkyl- or aralkyl-oxycarbonylation (typicallywith di-tert-butyl dicarbonate, benzyl chloroformate, or methylchloroformate and a base) or N-acylation (typically with trifluoroaceticanhydride and a base) to give a compound of formula (VII): ##STR9##[wherein R is an alkyl- or aralkyl-oxycarbonyl group or an optionallysubstituted alkyl- or aryl-carbonyl group and Z' is a hydroxy group]

which is then either:

(i) 5-O-sulfonylated (typically with p-toluenesulfonyl chloride,methanesulfonyl chloride or trifluoromethanesulfonic anhydride and abase) to give a compound of formula (VII) [wherein R is an alkyl- oraralkyl-oxycarbonyl group or an optionally substituted alkyl- oraryl-carbonyl group and Z' is an optionally substituted alkyl- oraryl-sulfonyloxy group], then subjected to a sulfonate displacementreaction with a reagent capable of providing a nucleophilic source ofhalide ion (typically sodium, lithium or a tetraalkylammonium fluoride,chloride, bromide, or iodide); or

(ii) subjected to a reagent system capable of directly replacing aprimary hydroxy group with a halogen atom, for example as in theMitsunobu reaction (e.g. using triphenylphosphine, diethylazodicarboxylate and a nucleophilic source of halide ion as above), byreaction with diethylaminosulfur trifluoride (DAST), or by reaction withmethyltriphenoxyphosphonium iodide in dimethylformamide [see e.g.Stoeckler et al, Cancer Res., 46 (1986) 1774] or by reaction withthionyl chloride or bromide in a polar solvent such ashexamethylphosphoramide [Kitagawa and Ichino, Tetrahedron Lett., (1971)87];

to give a compound of formula (VII) [wherein R is an alkyl- oraralkyl-oxycarbonyl group or an optionally substituted alkyl- oraryl-carbonyl group and Z' is a halogen atom], which is then selectivelyN-deprotected by acid- or alkali-catalyzed hydrolysis or alcoholysis orcatalytic hydrogenolysis as required for the N-protecting group in use.

A compound of formula (VII) [wherein R is an alkyl- oraralkyl-oxycarbonyl group or an optionally substituted alkyl- oraryl-carbonyl group and Z' is a hydroxy group] can also be prepared froma compound of formula (II) [wherein Z' is a trialkylsilyloxy,alkyldiarylsilyloxy or optionally substituted triarylmethoxy group], byN-alkyl- or aralkyl-carboxylation or N-acylation as above, thenselective 5-O-deprotection by acid-catalyzed hydrolysis or alcoholysis,catalytic hydrogenolysis, or treatment with a source of fluoride ion (egtetrabutylammonium fluoride) as required for the 5-O-protecting group inuse.

The compound of formula (II) [wherein Z' is a hydrogen atom] can beprepared from either:

(i) a 5-hydroxy compound of formula (VII) [wherein R is an alkyl- oraralkyl-oxycarbonyl group or an optionally substituted alkyl- oraryl-carbonyl group and Z' is a hydroxy group], by formation and radicaldeoxygenation of a 5-O-thioacyl derivative; or

(ii) a 5-deoxy-5-halogeno-compound of formula (VII) [wherein Z' is achlorine, bromine or iodine atom] by reduction, either using a hydridereagent such as tributyltin hydride under free radical conditions, or bycatalytic hydrogenolysis, typically with hydrogen over a palladiumcatalyst;

followed by selective N-deprotection by acid- or alkali-catalyzedhydrolysis or alcoholysis or catalytic hydrogenolysis as required forthe N-protecting group in use.

A compound of formula (II) [wherein Z' is an optionally substitutedalkylthio, aralkylthio or arylthio group] can be prepared by reaction ofa 5-deoxy-5-halogeno or a 5-O-sulfonate derivative of formula (VII)[wherein R is an alkyl- or aralkyl-oxycarbonyl group or an optionallysubstituted alkyl- or aryl-carbonyl group and Z' is a halogen atom or anoptionally substituted alkyl- or aryl-sulfonyloxy group] mentionedabove, with an alkali metal or tetraalkylammonium salt of thecorresponding optionally substituted alkylthiol, aralkylthiol orarylthiol followed by selective N-deprotection by acid- oralkali-catalyzed hydrolysis or alcoholysis or catalytic hydrogenolysisas required for the N-protecting group in use [see e.g. Montgomery etal., J. Med. Chem., 17 (1974) 1197].

The compound of formula (II) [wherein Z' is a group of formula OQ, and Qis an optionally substituted alkyl, aralkyl or aryl group] can beprepared from a 5-hydroxy compound of formula (VII) [wherein R is analky- or aralkyl-oxycarbonyl group or an optionally substituted alkyl-or aryl-carbonyl group and Z' is a hydroxy group], by

(i) reaction with an alkyl or aralkyl halide in the presence of a base(e.g. methyl iodide and sodium hydride, or benzyl bromide and sodiumhydride, in tetrahydrofuran as solvent); or

(ii) sequential conversion to a 5-O-sulfonate derivative (as above) andreaction with an alkali metal or tetraalkylammonium salt of the desiredphenol,

followed by selective N-deprotection by acid- or alkali-catalyzedhydrolysis or alcoholysis or catalytic hydrogenolysis as required forthe N-protecting group in use.

It will be appreciated that the conversions above are conventionalreactions employed in carbohydrate chemistry. Many alternative reagentsand reaction conditions can be employed that will effect theseconversions, and references to many of these can be found in theSpecialist Periodical Reports "Carbohydrate Chemistry", Volumes 1-28,published by the Royal Society of Chemistry, particularly in thechapters on Halogeno-sugars, Amino-sugars, Thio-sugars, Esters,Deoxy-sugars, and Nucleosides.

Method (B):

(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidines)

reacting a compound of formula (V) [wherein Z' is as defined for formula(II) where first shown above, and R is an alkyl group] with benzoylisothiocyanate then methyl iodide in the presence of a base (e.g. DBU orDBN) following the approach used to prepare 9-deazaguanosine and itsderivatives [see e.g. Montgomery et al., J. Med. Chem., 36 (1993) 55,Lim et al., J. Org. Chem., 48 (1983) 780, and references therein] togive a compound of formula (VIII) ##STR10## [wherein Z' is atrialkylsilyloxy, alkyldiarylsilyloxy or optionally substitutedtriarylmethoxy group, a hydrogen or halogen atom, SQ or OQ wherein Q isan optionally substituted alkyl, aralkyl or aryl group and R is an alkylgroup]

(typically Z', when a protected hydroxy group, is atert-butyldimethylsilyloxy, trityloxy or similar group, and R is amethyl or ethyl group)

which is then cyclized in the presence of ammonia to give a separablemixture of compounds of formula (IX) ##STR11## [wherein D is an amino ormethylthio group, and Z' and R are as defined for formula (VIII) wherefirst shown above, or Z' is a hydroxy group]

(where for example a tert-butyldimethylsilyloxy group has been cleavedunder the reaction conditions)

and the product of formula (IX) [wherein D is an amino or methylthiogroup] is fully deprotected under acidic conditions by the proceduresset out in Method (A).

Method (C):

(4-aminopyrrolo[3,2-d]pyrimidines)

reacting a compound of formula (IV) [wherein Z' is as defined forformula (II) where first shown above] with aminoacetonitrile undermildly basic conditions, cyclization of the product by reaction with asimple ester of chloroformic acid (typically benzyl chloroformate ormethyl chloroformate) to give a compound of formula (X) ##STR12##[wherein Z' is a trialkylsilyloxy, alkyldiarylsilyloxy or optionallysubstituted triarylmethoxy group, a hydrogen or halogen atom, SQ or OQwherein Q is an optionally substituted alkyl, aralkyl or aryl group andR is an aralkyl or alkyl group]

(typically Z', when a protected hydroxy group, is atert-butyldimethylsilyloxy, trityloxy or similar group, and R is abenzyl or methyl group)

which is then deprotected on the pyrrole nitrogen by hydrogenolysis inthe presence of a noble metal catalyst (e.g. Pd/C) in the case of abenzyl group or under mildly basic conditions in the case of a simplealkyl group such as a methyl group, and processed as described above forthe transformation (V) →(VI)→(I) or (V)→(VIII)→(IX)→(I). This methodfollows the approach used to prepare 9-deazaadenosine and its analogues[Lim and Klein, Tetrahedron Lett., 22 (1981) 25, and Xiang et al.,Nucleosides Nucleotides 15 (1996) 1821]

Method (D):

(7-hydroxypyrazolo[4,3-d]pyrimidines--Daves' methodology)

reacting a compound of formula (II) [as defined where first shown above]sequentially with N-chlorosuccinimide and a hindered base (such aslithium tetramethylpiperidide) to form an imine, then condensing thiswith the anion produced by abstraction of the bromine or iodine atomfrom a compound of formula (XIa) or (XIb) ##STR13## [wherein R³ is abromine or iodine atom and R⁴ is a tetrahydropyran-2-yl group]

typically using butyllithium or magnesium, to give a product which isthen fully deprotected under acidic conditions (as in Method A). Methodsfor preparing compounds of formula (XIa) and (XIb) and mixtures thereofare described in Zhang and Daves, J. Org. Chem., 57 (1992) 4690, Stoneet al., J. Org. Chem., 44 (1979) 505, and references therein.

It will be appreciated that while the tetrahydropyran-2-yl group isfavoured as the protecting group for this reaction, other O,N-protectinggroups can be used, and that this method will also be applicable to thesynthesis of analogous pyrazolo[4,3-d]pyrimidines bearing substituentsat position-5 and/or -7 of the pyrazolo[4,3-d]pyrimidine ringindependently chosen from a hydroxy group, an amino, alkylamino, oraralkylamino group or a hydrogen atom using analogues of compounds offormula (XIa) and (XIb) in which the ionizable hydrogen atoms of anyhydroxy or amino groups have been replaced by a suitable protectinggroups.

Method (E):

(7-hydroxypyrazolo[4,3-d]pyrimidines--Yokoyama method)

subjecting a 5-O-ether protected 2,3-O-isopropylidene-D-ribofuranosederivative, where the 5-ether substituent is typically a trialkylsilyl,alkyldiarylsilyl, an optionally substituted triarylmethyl or anoptionally substituted aralkyl group, particularly atert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilyl,trityl or benzyl group, to the following reaction sequence:

(i) condensation with the anion produced by abstraction of the bromineor iodine atom from a compound of formula (XIa) or (XIb) from Method(D);

(ii) oxidation of the resulting diol to a diketone, typically using aSwern oxidation or a variant thereof using a dimethylsulfoside-basedoxidant (e.g. using a dimethylsulfoxide and trifluoroacetic anhydridereagent combination in dichloromethane solution at low temperature,typically -78° C., followed by triethylamine and warming to roomtemperature);

(iii) double reductive amination to form a1,4-dideoxy-1,4-imino-D-ribitol moiety, typically with sodiumcyanoborohydride and ammonium formate, ammonium acetate orbenzhydrylamine in methanol; and

(iv) removal of the protecting groups by acid-catalyzed hydrolysis (e.g.with 70% aqueous trifluoroacetic acid) and if required (as in the caseof the product made with benzhydrylamine or where an optionallysubstituted aralkyl group has been used for protecting the primaryhydroxyl group in the iminoribitol moiety) hydrogenolysis over a metalcatalyst (typically a palladium catalyst) or if desired (as in the caseof silyl ether protecting group) exposure to a reagent capable of actingas a source of fluoride ion, e.g. tetrabutylammonium fluoride intetrahydrofuran or ammonium fluoride in methanol). Conditions suitablefor effecting this sequence of reactions are reported in Yokoyama etal., J. Org. Chem., 61 (1996) 6079, and conditions for double reductiveamination with ammonium acetate or benzhydrylamine can be found inFurneaux et al., Tetrahedron 42 (1993) 9605 and references therein.

Method (F):

(7-hydroxypyrazolo[4,3-d]pyrimidines--the Kalvoda method)

reacting a compound of formula (II) [as defined where first shown above]sequentially with N-chlorosuccinimide and a hindered base (such aslithium tetramethylpiperadide) to form an imine, then with a combinationof trimethylsilyl cyanide and a Lewis acid (typically boron trifluoridediethyl etherate) followed by acid catalyzed hydrolysis to give acompound of formula (XII) ##STR14## [wherein Z' is a hydrogen or halogenatom, a hydroxy group, or a group of formula SQ or OQ where Q is anoptionally substituted alkyl, aralkyl or aryl group]

which is then converted by sequential selective N-protection (typicallywith trifluoroacetic anhydride, di-tert-butyl dicarbonate, benzylchloroformate, or methyl chloroformate and a base), and O-protectionwith an acyl chloride or anhydride and a base (typically aceticanhydride or benzoyl chloride in pyridine) to a suitably protectedderivative of formula (XIII) ##STR15## [wherein R¹ is an alkyl- oraralkyl-oxycarbonyl group or an optionally substituted alkyl- oraryl-carbonyl group, Z' is a hydrogen or a halogen atom, a group offormula SQ or OQ where Q is an optionally substituted alkyl, aralkyl oraryl group, or a group of formula R² O, and R² is an alkylcarbonyl oroptionally substituted arylcarbonyl group]

(typically R¹ will be a trifluoroacetyl, tert-butoxycarbonyl orbenzyloxycarbonyl group, and R² will be an acetyl or benzoyl group).

The carboxylic acid moiety In the resulting compound of formula (XIII)is then transformed into a pyrazolo[4,3-d]pyrimidin-7-one-3-yl moietyfollowing the method described by Kalvoda [Collect. Czech. Chem.Commun., 43 (1978) 1431], by the following sequence of reactions:

(i) chlorination of the carboxylic acid moiety to form an acyl chloride,typically with thionyl chloride with a catalytic amount ofdimethylformamide in an inert solvent;

(ii) use of the resulting acyl chloride to acylate hydrogen cyanide inthe presence of tert-butoxycarbonyltriphenylphosphorane (i.e. Ph₃P=CHCO₂ Bu^(t)) to give a 3-cyano-2-propenoate derivative;

(iii) cycloaddition of this with diazoacetonitrile (which can beprepared from aminoacetonitrile hydrochloride and sodium nitrite) withconcomitant elimination of hydrogen cyanide to give a pyrazolederivative;

(iv) acid-catalyzed hydrolysis of the tert-butyl ester in this pyrazolederivative to its equivalent carboxylic acid;

(v) Curtius reaction, typically with phenylphosphoryl azide and2,2,2-trichloroethanol in the presence of triethylamine, which convertsthe carboxylic acid moiety into a 2,2,2-trichloroethoxycarbonylaminogroup (i.e. the product is a carbamate);

(vi) reductive cleavage of this trichloroethyl carbamate, typically withzinc dust in methanol containing ammonium chloride;

(vii) condensation of the resulting ethyl3-amino-5-substituted-pyrazole-2-carboxylate with formamidine acetate togive a compound of formula (XIV) ##STR16## [wherein R¹ is an alkyl- oraralkyl-oxycarbonyl group or an optionally substituted alkyl- oraryl-carbonyl group, Z' is a hydrogen or a halogen atom, SQ or OQ whereQ is an optionally substituted alkyl, aralkyl or aryl group, or a groupof formula R² O, and R² is an alkylcarbonyl or optionally substitutedarylcarbonyl group, A is a nitrogen atom, B is a hydroxy group and D isa hydrogen atom]

which is then N- and O-deprotected by acid- or alkali-catalyzedhydrolysis or alcoholysis or catalytic hydrogenolysis as required forthe O- and N-protecting groups in use.

Method (G):

(7-aminopyrazolo 4,3-d]pyrimidines--the Buchanan method)

reacting a compound of formula (II) [as defined where first shown above]sequentially with N-chlorosuccinimide and a hindered base (such aslithium tetramethylpiperadide) to form an imine, which is thentransformed into a 7-amino-pyrazolo[4,3-d]pyrimidine derivativefollowing the approach used to prepare formycin and its analogues byBuchanan and co-workers [J. Chem. Soc., Perkin Trans. I (1991) 1077 andreferences therein], by the following sequence of reactions:

(i) addition of 3,3-diethoxyprop-1-ynylmagnesium bromide or3,3-diethoxyprop-1-ynyllithium to the imine;

(ii) N-protection, typically with trifluoroacetic anhydride,di-tert-butyl dicarbonate, benzyl chloroformate, or methyl chloroformateand a base;

(iii) mild acid hydrolysis to remove the acid sensitive O-protectinggroups and convert the diethyl acetal moiety into an aldehydic moiety;

(iv) condensation with hydrazine to convert the 3-substitutedprop-2-ynal derivative into a 3-substituted pyrazole derivative;

(v) acylation, typically with acetic anhydride or benzoyl chloride inpyridine;

(vi) nitration, typically with ammonium nitrate, trifluoroaceticanhydride and trifluoroacetic acid, to produce an 3-substituted1,4-dinitopyrazole derivative;

(vii) reaction with a reagent capable of delivering cyanide ion,typically sodium cyanide in aqueous ethanol to cause a cine-substitutionof one of the two nitro-groups;

(viii) reduction of the residual nitro-group, typically with sodiumdithionite or by catalytic hydrogenation over a metal catalyst;

(ix) condensation with formamidine acetate to give a compound of formula(XIV) [wherein R¹ is an alkyl- or aralkyl-oxycarbonyl group or anoptionally substituted alkyl- or aryl-carbonyl group, Z' is a hydrogenor a halogen atom, SQ or OQ where Q is an optionally substituted alkyl,aralkyl or aryl group, or a group of formula R² O wherein R² is analkylcarbonyl or optionally substituted arylcarbonyl group, A is anitrogen atom, B is an amino group and D is a hydrogen atom]

which is then N- and O-deprotected by acid- or alkali-catalyzedhydrolysis or alcoholysis or catalytic hydrogenolysis as required forthe O- and N-protecting groups in use.

Method (H):

(2'-deoxy-analogues)

effecting the overall 2'-deoxygenation of a compound of formula (I)[wherein X and Z are hydroxy groups, Y is a hydrogen atom, and A, B andD are as defined where this formula Is first shown above] throughsequential:

(i) selective N-alkyl- or aralkyl-oxycarbonylation (typically withdi-tert-butyl dicarbonate, benzyl chloroformate, or methyl chloroformateand a base) or N-acylation (typically with trifluoroacetic anhydride anda base) of the 1,4-dideoxy-1,4-iminoribitol moiety in such a compound offormula (I); and

(ii) 3',5'-O-protection of the resulting product by reaction with1,3-dichloro-1,1,3,3-tetraisopropyldislloxane and a base to give acompound of formula (XV): ##STR17## [wherein R¹ is an alkyl- oraralkyl-oxycarbonyl group or an optionally substituted alkyl- oraryl-carbonyl group, R² is either the same as R¹ or is a hydrogen atom,and A, B and D are as defined for formula (I) where first shown above]

(iii) 2'-O-thioacylation of the resulting compound of formula (XV)(typically with phenoxythionocarbonyl chloride and a base; or sodiumhydride, carbon disulfide and methyl iodide);

(iv) Barton radical deoxygenation (typically with tributyltin hydrideand a radical initiator);

(v) cleavage of the silyl protecting group by a reagent capable ofacting as a source of fluoride ion, e.g. tetrabutylammonium fluoride intetrahydrofuran or ammonium fluoride in methanol; and

(vi) cleavage of the residual N- and O-protecting groups by acid- oralkali-catalyzed hydrolysis or alcoholysis or catalytic hydrogenolysisas required for the protecting groups in use.

Reagents and reaction conditions suitable for conducting the key stepsin this transformation can be found in Robins et al., J. Am. Chem. Soc.,105 (1983) 4059; Solan and Rosowsky, Nucleosides Nucleotides 8 (1989)1369; and Upadhya et al., Nucleic Acid Res., 14(1986) 1747.

It will be appreciated that a compound of formula (I) has a nitrogenatom in its pyrrole or pyrazole ring capable of undergoing alkyl- oraralkyl-oxycarbonylation or acylation during step (i), or thioacylationduring step (ii), depending upon the reaction conditions employed.Should such derivatives be formed, the pyrrole or pyrazoleN-substituents in the resulting derivatives are either sufficientlylabile that they can be removed by mild acid- or alkali-catalyzedhydrolysis or alcoholysis, or do not interfere with the subsequentchemistry in the imino-ribitol moiety, and can be removed during thefinal deprotection step(s). If desired, this approach can be applied toa compound of formula (XV) [as defined above, but additionally bearingN-protecting groups on the pyrazolo- or pyrrolo-pyrimidine moiety].Methods suitable for preparing such N-protected compounds can be foundin Ciszewski et al., Nucleosides Nucleotides 12 (1993) 487; andKambhampati et al., Nucleosides and Nucleotides 5 (1986) 539, as canmethods to effect their 2'-deoxygenation, and conditions suitable forN-deprotection.

Method (I):

(2'-epi-analogues)

effecting the overall C-2' epimerization of a compound of formula (I),by oxidizing and then reducing a compound of formula (XV) [as definedwhere first shown above] to give compound of formula (XVI): ##STR18##[wherein R¹, R², A, B and D are as defined for formula (XV) where firstshown above]

which may be present in a mixture with the starting alcohol of formula(XV), and then fully deprotecting this compound of formula (XVI) as setout in steps (v) and (vi) of Method H.

Reagents and reaction conditions suitable for conducting the key stepsin this transformation can be found in Robins et al., Tetrahedron 53(1997) 447.

Method (J):

(2'-deoxy-2'-halogeno- and 2'-deoxy-2'-epi-2'-halogeno-analogues)

reacting a compound of formula (XV) or (XVI) [as defined where firstshown above] by the methods set out in Method (A) for the preparation ofa compound of formula (II) [wherein Z' is a halogen atom] which involveeither

(i) 2'-O-sulfonylation and sulfonate displacement with a halide ion; or

(ii) direct replacement of the 2'-hydroxy group with a halogen atom,e.g. by the Mitsunobu reaction or reaction with diethylaminosulfurtrifluoride (DAST)

to give a compound of inverted stereochemistry at C-2', which is thenfully deprotected as set out in steps (v) and (vi) of Method H.

It will be appreciated that a compound of formula (XV) or (XVI) has anitrogen atom in its pyrrole or pyrazole ring capable of undergoingsulfonylation during step (i), depending upon the reaction conditionsemployed. Should such derivatives be formed, the pyrrole or pyrazoleN-sulfonate substituents in the resulting derivatives are eithersufficiently labile that they can be removed by mild acid- oralkali-catalyzed hydrolysis or alcoholysis, or do not interfere with thesubsequent chemistry in the iminoribitol moiety, and can be removedduring the final deprotection step(s).

If desired, this approach can be applied to a compound of formula (XV)or (XVI) [as defined above, but additionally bearing N-protecting groupson the pyrazolo- or pyrrolo-pyrimidine moiety]. Methods suitable forpreparing such N-protected compounds can be found in Ciszewski et al.,Nucleosides Nucleotides 12 (1993) 487; and Kambhampati et al.,Nucleosides and Nucleotides 5 (1986) 539, as can methods to effect2'-O-triflate formation and displacement by halide ion with inversion,and conditions suitable for N-deprotection.

Method (K):

(5'-deoxy-, 5'-deoxy-5'-halogeno-, 5'-ether and 5'-thio-analogues)

by applying the procedures described in Method (A) for converting acompound of formula (VII) [wherein R is an alkyl- or aralkyl-oxycarbonylgroup or an optionally substituted alkyl- or aryl-carbonyl group and Z'is a hydroxy group] into a compound of formula (II) [wherein Z' is ahalogen or hydrogen atom or SQ or OQ where Q is an optionallysubstituted alkyl, aralkyl or aryl group alkylthio group of one to fivecarbon atoms] to a compound of formula (XVII): ##STR19## (wherein R isan alkyl- or aralkyl-oxycarbonyl group or an optionally substitutedalkyl- or aryl-carbonyl group, Z' is a hydroxy group, and A, B and D areas defined for formula (I) where first shown above). which is then fullydeprotected under acidic conditions, e.g. by treatment with aqueoustrifluoroacetic acid.

Such a compound of formula (XVII) can be prepared from a compound offormula (I) [wherein X and Z are both hydroxy groups, Y is a hydrogenatom and A, B, and D have the meanings defined for formula (I) wherefirst shown above] in the following two reaction steps, which may beapplied in either order:

(i) selective N-alkyl- or aralkyl-oxycarbonylation (typically withdi-tert-butyl dicarbonate, benzyl chloroformate, or methyl chloroformateand a base) or N-acylation (typically with trifluoroacetic anhydride anda base) of the 1,4-dideoxy-1,4-iminoribitol moiety; and

(ii) 2',3'-O-isopropylidenation, which may be effected with a variety ofreagents, e.g. acetone and anhydrous copper sulfate with or withoutadded sulfuric acid; acetone and sulfuric acid; 2,2-dimethoxypropane andan acid catalyst; or 2-methoxypropene and an acid catalyst.

It will be appreciated that such a compound of formula (I) or formula(XVII) has a nitrogen atom in its pyrrole or pyrazole ring capable ofundergoing sulfonylation, thioacylation, acylation oraralkyl-oxycarbonylation, depending upon the reaction conditionsemployed. Should such derivatives be formed, the pyrrole or pyrazoleN-substituents in the resulting derivatives are either sufficientlylabile that they can be removed by mild acid- or alkali-catalyzedhydrolysis or alcoholysis, or do not interfere with the subsequentchemistry in the iminoribitol moiety, and can be removed during thefinal deprotection step(s).

Method (L):

(2- and 4-aminopyrrolo[3,2-d]pyrimidine and 5- and7-aminopyrazolo[4,3-d]pyrimidine analogues)

chlorinating a compound of formula (XVIII) ##STR20## [wherein

R¹ is an alkyl- or aralkyl-oxycarbonyl group or an optionallysubstituted alkyl- or aryl-carbonyl group,

R² is an alkylcarbonyl or optionally substituted arylcarbonyl group,

X and Y are independently chosen from a hydrogen or halogen atom, or agroup of formula R² O, except that when one of X or Y is a halogen atomor a group of formula R² O, the other is a hydrogen atom,

Z' is a group of formula R² O or, when X is a group of formula R² O, Z'is a hydrogen or halogen atom, a group of formula R² O or of formula OQor SQ wherein Q is an optionally substituted alkyl, aralkyl or an arylgroup,

A is a nitrogen atom or a methine group, and one of B or D is a hydroxygroup, and the other is a chlorine, bromine or hydrogen atom]

with a chlorinating reagent, and then displacing the chlorine atom witha nitrogen nucleophile by one of the following methods:

(i) ammoniolysis, typically using liquid ammonia, concentrated aqueousammonia, or a solution of ammonia in an alcohol such as methanol; or

(ii) conversion first to a triazole derivative, by addition of4-chlorophenyl phosphorodichloridate to a solution of the chloride and1,2,4-triazole in pyridine, and alkaline hydrolysis of both thetetrazole moiety and the ester protecting groups with ammoniumhydroxide;

(iii) reaction with a source of azide ion, e.g. an alkali metal azide ortetraalkylammonium azide, and reduction of the resulting product,typically by catalytic hydrogenation; or

(iv) reaction with an alkylamine or aralkylamine, such as methylamine orbenzylamine in methanol.

These conditions are sufficiently basic that O-ester groups willgenerally be cleaved but any residual O- or N-protecting groups can thenbe removed by acid- or alkali-catalyzed hydrolysis or alcoholysis orcatalytic hydrogenolysis as required for the protecting groups in use.

Suitable chlorinating agents are thionyl chloride--dimethylformamidecomplex [Ikehara and Uno, Chem. Pharm. Bull., 13 (1965) 221],triphenylphosphine in carbon tetrachloride and dichloromethane with orwithout added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) [De Napoli etal., J. Chem. Soc., Perkin Trans.1 (1995) 15 and references therein],phosphoryl chloride [Imai, Chem. Pharm. Bull., 12 (1964) 1030], orphenylphosphoryl chloride and sodium hydride.

Suitable conditions for such an ammoniolysis or a reaction with analkylamine can be found in Ikehara and Uno, Chem. Pharm. Bull., 13(1965) 221; Robins and Tripp, Biochemistry 12 (1973) 2179; Marumoto etal., Chem. Pharm. Bull., 23 (1975) 759; and Hutchinson et al., J. Med.Chem., 33 (1990) 1919].

Suitable conditions for conversion of a such a chloride to an amine viaa tetrazole derivative can be found in Lin et al., Tetrahedron 51 (1995)1055.

Suitable conditions for reaction with azide ion followed by reductioncan be found in Marumoto et al., Chem. Pharm. Bull., 23 (1975) 759.

Such a compound of formula (XVIII) can be prepared from a compound offormula (I) by selective N-alkyl- or aralkyl-oxycarbonylation (typicallywith di-tert-butyl dicarbonate, benzyl chloroformate, or methylchloroformate and a base) or N-acylation of the1,4-dideoxy-1,4-iminoribitol moiety and then O-acylation (typically withacetic anhydride or benzoyl chloride in pyridine). It will beappreciated that such a compound of formula (I) has a nitrogen atom inits pyrrole or pyrazole ring capable of undergoing alkyl- oraralkyl-oxycarbonylation or acylation depending upon the reactionconditions employed. Should such derivatives be formed, the pyrrole orpyrazole N-substituents in the resulting derivatives are eithersufficiently labile that they can be removed by mild acid- oralkali-catalyzed hydrolysis or alcoholysis, or do not interfere with thesubsequent chemistry, and can be removed during the final deprotectionstep(s).

The above chlorination--amination--deprotection sequence can also beapplied to a compound of formula (XVII) [wherein B is a hydroxy group, Dis a hydrogen atom, Z' is a hydrogen or halogen atom, or a group offormula R² O, R² is a trialkylsilyloxy or alkyldiarylsilyloxy group, oran optionally substituted triarylmethoxy, alkylcarbonyl or arylcarbonylgroup, R and A are as defined for formula (XVII) where first shownabove]. Suitable conditions for conducting this reaction sequence can befound in Ikehara et al., Chem. Pharm. Bull., 12 (1964) 267.

Method (M):

(2,4-dihydroxypyrrolo[3,2-d]pyrimidine and5,7-dihydroxypyrazolo[4,3-d]pyrimidine analogues)

oxidation of either:

(i) a compound of formula (XVIII) [wherein R² is a hydrogen atom; X andY are independently chosen from a hydrogen or halogen atom, or a hydroxygroup, except that when one of X or Y is a halogen atom or a hydroxygroup, the other is a hydrogen atom; Z' is a hydroxy group or, when X isa hydroxy group, Z' is a hydrogen or halogen atom, a hydroxy group, orOQ; Q is an optionally substituted alkyl, aralkyl or aryl group; B is ahydroxy group or an amino group; D is a hydrogen atom; and R¹ and A areas defined for formula (XVIII) where first shown above] with bromine inwater; or

(ii) a compound of formula (XVIII) [wherein Z' is a hydrogen or ahalogen atom, or a group of formula R² O, or OQ; Q is an optionallysubstituted alkyl, aralkyl or aryl group; B is a hydroxy group or anamino group, D is a hydrogen atom and R¹, R², X, Y and A are as definedfor formula (XVIII) where first shown above], with bromine or potassiumpermanganate in water or in an aqueous solvent mixture containing aninert, water-miscible solvent to improve the solubility of thesubstrate, to give a related compound of formula (XVIII) [but wherein Band D are now hydroxy groups], and then

removal of any O- and N-protecting groups by acid- or alkali-catalyzedhydrolysis or alcoholysis or catalytic hydrogenolysis as required forthe protecting groups in use.

Such a compound of formula (XVIII) required for step (i) above can beprepared from a compound of formula (I) [wherein Z is Z', and X, Y, Z,A, B and D are as defined for the required compound of formula (XVIII)]by selective N-alkyl- or aralkyl-oxycarbonylation (typically withdi-tert-butyl dicarbonate, benzyl chloroformate, or methyl chloroformateand a base) or N-acylation (typically with trifluoroacetic anhydride anda base) of the 1,4-dideoxy-1,4-iminoribitol moiety. This can then beconverted to the corresponding compound of formula (XVIII) required forstep (ii) above by O-acylation (typically with acetic anhydride orbenzoyl chloride in pyridine). It will be appreciated that such acompound of formula (I) has a nitrogen atom in its pyrrole or pyrazolering capable of undergoing alkyl- or aralkyl-oxycarbonylation oracylation depending upon the reaction conditions employed. Should suchderivatives be formed, the pyrrole or pyrazole N-substituents in theresulting derivatives are either sufficiently labile that they can beremoved by mild acid- or alkali-catalyzed hydrolysis or alcoholysis, ordo not interfere with the subsequent chemistry, and can be removedduring the final deprotection step(s).

Method (N):

(4-amino-2-chloropyrrolo[3,2-d]pyrimidine and7-amino-5-chloropyrazolo[4,3-d]pyrimidine analogues)

chlorinating a compound of formula (XVIII) [wherein B and D are hydroxygroups and R¹, R², X, Y, Z' and A are as defined for formula (XVIII)where first shown above] to give a corresponding dichloro-derivative offormula (XVIII) [wherein B and D are chlorine atoms], typically withneat phosphorous oxychloride, and then displacing the more reactivechloro-substituent selectively by ammoniolysis, typically usinganhydrous liquid ammonia in a pressure bomb or methanolic ammonia, whichsimultaneously cleaves the O-ester protecting groups. The residualN-protecting group is then removed by acid-catalyzed hydrolysis oralcoholysis or catalytic hydrogenolysis as required for the protectinggroups in use, to give a compound of formula (I) [wherein B is anamino-group and D is a chlorine atom].

The above dichloro-derivative of formula (XVIII) can be converted into acompound of formula (I) [wherein B and D are chlorine atoms] by removalof the O- and N-protecting groups by acid- or alkali-catalyzedhydrolysis or alcoholysis as required for the protecting groups in use.It will be appreciated that one of the chlorine atoms in theaforementioned compound of formula (XVIII) or of formula (I) is quitereactive and that conditions chosen for deprotection must be mild enoughthat they limit unwanted reactions involving this atom.

Suitable reaction conditions for the key steps in this method can befound in Upadhya et al., Nucleic Acid Res., 14 (1986) 1747 and Kitagawaet al., J. Med. Chem., 16 (1973) 1381.

Method (O):

(2-chloro-4-hydroxypyrrolo[3,2-d]pyrimidine and5-chloro-7-hydroxypyrazolo[4,3-d]pyrimidine analogues fromdichloro-compounds)

hydrolysis of a compound of formula (XVIII) [wherein B and D arechlorine atoms] available as an intermediate from the first reaction ofMethod N, typically with aqueous potassium hydroxide or sodiumcarbonate, in the presence of an inert, water-miscible solvent such asdioxane to enhance solubility as required, followed by removal of theresidual N-protecting group by acid-catalyzed hydrolysis or alcoholysisor catalytic hydrogenolysis as required for the protecting groups inuse, to give a compound of formula (I) [wherein B is a hydroxy group andD is a chlorine atom].

Method (P):

(2-chloro-4-hydroxypyrrolo[3,2-d]pyrimidine and5-chloro-7-hydroxypyrazolo[4,3-d]pyrimidine analogues fromaminochloro-compounds)

deamination of a compound of formula (XVIII) [wherein B is an aminogroup, D is a chlorine atom, R¹ is an alkyl- or aralkyl-oxycarbonylgroup or an optionally substituted alkyl- or aryl-carbonyl group, R² isa hydrogen atom, Z'=Z and X, Y, Z and A are as defined for formula (I)where first shown above], available as an intermediate following thechlorination and ammonyolysis reactions of Method N, by reaction withnitrosyl chloride, followed by removal of the protecting groups as setout in Method N Typical reaction conditions can be found in Sanghvi etal., Nucleosides Nucleotides 10 (1991) 1417.

Method (Q):

(4-halogenopyrrolo[3,2-d]pyrimidine and7-halogenopyrazolo[4,3-d]pyrimidine analogues)

reacting a compound of formula (XVIII) [wherein R¹ istert-butoxycarbonyl group, B is a hydroxy group, D is a hydrogen atomand R², X, Y, Z' and A are as defined for formula (XVIII) where firstshown above] by a method used to prepare halogeno-formycin analogues[Watanabe et al., J. Antibiotic, Ser. A 19 (1966) 93] which involvessequential treatment with:

(i) phosphorous pentasulfide by heating in pyridine and water underreflux to give a mercapto-derivative;

(ii) methyl iodide to give a methylthio-derivative;

(iii) a base in a simple alcohol or an aqueous solution of a simplealcohol, e.g. sodium methoxide in methanol, to remove the O-protectinggroups; and

(iv) chlorine, bromine or iodine in absolute methanol to give ahalogeno-derivative

which is then N-deprotected by reaction with aqueous acid, typically aconcentrated trifluoroacetic acid solution.

Method (R):

(pyrrolo[3,2-d]pyrimidine and pyrazolo[4,3-dipyrimidine analogues)

hydrogenolytic cleavage of the chloride intermediate resulting from thechlorination reaction used as the first reaction in Method L, or thechloride intermediate resulting from the chlorination reaction step (iv)in Method Q, or the compound of formula (I) produced by Method Q,typically using hydrogen over palladium on charcoal as the catalyst,optionally with magnesium oxide present to neutralize released acid,followed by cleavage of any residual O- or N-protecting groups by acid-or alkali-catalyzed hydrolysis or alcoholysis as required for theprotecting groups in use.

Method (S):

(N-alkylated 4-aminopyrrolo[3,2-d]pyrimidine and7-aminopyrazolo[4,3-d]pyrimidine analogues)

heating an O-deprotected methylthio-derivative produced by step (iii) ofMethod (Q) with an amine, e.g. methylamine, in absolute methanol in asealed tube or bomb, and then removing the N-protecting group byreaction with aqueous acid, typically a concentrated trifluoroaceticacid solution. This method has been used to prepare N-alkylated-formycinanalogues [Watanabe et al., J. Antibiotic, Ser. A 19 (1966) 93]; or

reacting a compound of formula (I) [wherein either B or D is an aminogroup] with 1,2-bis[(dimethylamino)methylene]hydrazine andtrimethylsilyl chloride in toluene to convert the amino group into a1,3,4-triazole group, hydrolysis to cleave the O-silyl groups (e.g. withacetic acid in aqueous acetonitrile), and displacement of the1,3,4-triazole group with an alkylamine in a polar solvent (e.g. wateror aqueous pyridine). This method has been used to prepareN,N-dimethyl-formycin A [Miles et al., J. Am. Chem. Soc., 117 (1995)5951]; or

subjecting a compound of formula (I) [wherein either B or D is an aminogroup] to an exchange reaction by heating it with an excess of analkylamine. This method has been used to prepare N-alkyl-formycin Aderivatives [Hecht et al., J. Biol. Chem., 250 (1975) 7343].

Method T:

(2-chloro-4-hydroxypyrrolo[3,2-d]pyrimidine and5-chloro-7-hydroxypyrazolo[4,3-d]pyrimidine analogues)

Selective chlorination of dihydroxy compound of formula (XVIII) [whereinB and D are hydroxy groups, and R¹, R², X, Y, Z' and A are as definedfor formula (XVIII) where first shown above], taking advantage of thegreater reactivity of the 4-hydroxy group on a2,4-dihydroxypyrrolo[3,2-d]pyrimidine derivative and the 7-hydroxy groupon a 5,7-dihydroxypyrazolo[4,3-d]pyrimidine derivative, followed byremoval of protecting groups, using the methods set out in Method N.

Method U:

(2-halogeno-, 4-halogeno- and 2,4-dihalogeno-pyrrolo[3,2-d]pyrimidineand 5-halogeno-, 7-halogeno-, and5,7-dihalogeno-pyrazolo[4,3-d]pyrimidine analogues)

diazotization of a compound of formula (XVIII) [wherein one of B or D isan amino group, and the other is independently chosen from an aminogroup, or a halogeno or hydrogen atom, and R¹, R² , X, Y, Z' and A areas defined for formula (XVIII) where first shown above] and subsequentreaction using one of the following procedures:

(i) with nitrous acid (made in situ from sodium nitrite) in the presenceof a source of halide ion. For replacement of an amino-group with afluorine atom, a concentrated aqueous solution of fluoroboric acid[Gerster and Robins, J. Org. Chem., 31 (1966) 3258; Montgomery andHewson, J. Org. Chem., 33 (1968) 432] or hydrogen fluoride and pyridineat low temperature (e.g. -25 to -30° C.) [Secrist et al., J. Med. Chem.,29 (1986) 2069] can serve both as the mineral acid and the fluoride ionsource; or

(ii) with an alkyl nitrite, typically tert-butyl or n-butyl nitrite, ina non-aqueous solvent in the presence of a source of halide ion. Forreplacement of an amino-group with a chlorine atom, a combination ofchlorine and cuprous chloride, or antimony trichloride can be used inchloroform as solvent [Niiya et al, J. Med. Chem., 35 (1992) 4557 andreferences therein]; or

(iii) with an alkyl nitrite, typically tert-butyl or n-butyl nitrite, ina non-aqueous solvent coupled with photohalogenation. For replacement ofan amino group with a chlorine, bromine or iodine atom, carbontetrachloride, bromoform, or diiodomethane have been used as reagent andsolvent and an incandescent light source (e.g. a 200 W bulb) has beenused to effect photohalogenation Ford et al., J. Med. Chem., 38 (1995)1189; Driscoll et al., J. Med. Chem., 39 (1996) 1619; and referencestherein];

to give a corresponding compound of formula (XVIII) [wherein B is ahalogen atom and D is either a halogen atom or an amino group], followedby removal of the protecting groups as set out in Method N.

The same transformations can be effected for a corresponding startingcompound of formula (XVIII) [wherein one of B or D is an amino group,and the other is a hydroxy group] if the hydroxy group is firstconverted to a thiol group [Gerster and Robins, J. Org. Chem., 31 (1966)3258]. This conversion can be effected by reaction with phosphorouspentasulfide by heating in pyridine and water under reflux (see MethodQ).

Method V:

(4-iodo-pyrazolo[3,2-d]pyrimidine and 7-iodopyrazolo[4,3-djpyrimidineanalogues)

treatment of corresponding chloro-analogue of formula (I) [wherein B isa chlorine atom] with concentrated aqueous hydroiodic acid, followingthe method of Gerster et al., J. Org. Chem., 28 (1963) 945.

Method W:

(5'-deoxy-5'-halogeno- and 5'-thio-analogues)

by reacting a compound of formula (XVIII) [wherein R² is a hydrogenatom; X and Y are independently chosen from a hydrogen or halogen atom,or a hydroxy group, except that when one of X or Y is a halogen atom ora hydroxy group, the other is a hydrogen atom; Z' is a hydroxy group;and R¹, A, B and D are as defined for formula (XVIII) where first shownabove] with either

(i) a trisubstituted phosphine and a disulfide, e.g. tributylphosphineand diphenyl disulfide; or

(ii) a trisubstituted phosphine (e.g. triphenylphosphine) and carbontetrabromide; or

(iii) thionyl chloride or bromide.

and then removal of the N-protecting group by acid- or alkali-catalyzedhydrolysis or alcoholysis or catalytic hydrogenolysis as required forthe protecting group in use.

Conditions suitable for conducting such selective replacements of a5'-hydroxy group with a thio group or a halogen atom can be found inChern et al., J. Med. Chem., 36 (1993) 1024; and Chu et al., NucleosideNucleotides 5 (1986) 185.

FURTHER METHODS

Compounds of the invention may also be prepared by other methods as willbe apparent to those skilled in the art.

FURTHER ASPECTS

The compounds of the invention are useful both in free base form and inthe form of salts. The term "pharmaceutically acceptable salts" isintended to apply to non-toxic salts derived from inorganic or organicacids including for example salts derived from the following acids-hydrochloric, sulfuric, phosphoric, acetic, lactic, fumaric, succinic,tartaric, gluconic, citric, methanesulphonic and p-toluenesulphonicacids.

The compounds of the invention are potent inhibitors of purinenucleoside phosphorylases and/or nucleoside hydrolases. For example theIC₅₀ values for the compounds of formula (Ia) and formula (Ib) are lessthan 0.1 nM for both calf spleen PNP and human red blood cell PNP. Theexamples below provide further detail of the effectiveness of thisinhibitor. Purine nucleoside phosphorylase inhibitory activity can bedetermined by the coupled xanthine oxidase method using inosine as asubstrate (H. M. Kalckar, J.) Biol. Chem. 167 (1947) 429-443. Slow onsetinhibitor binding can be determined using methods such as thosedescribed by Merkler et al., Biochemistry 29 (1990) 8358-64. Parasitenucleoside hydrolase activity may be measured inter alia by methodsdisclosed in published PCT international patent application W097/31008and the references cited therein.

The potency of the inhibitors of the invention provides importantadvantages over the prior art because of the relatively high activity ofPNP in blood and mammalian tissue. As mentioned above the requireddosage of 9-(3-pyridylmethyl)-9-deazaguanine may be of the order of 3.5grams per dose for a human adult. The present invention provides theadvantage that considerably lower quantities of the compounds arerequired. This allows cost saving and may also reduce unwanted sideeffects.

The amount of active ingredient to be administered can vary widelyaccording to the nature of the patients and the nature and extent of thedisorder being treated. Typically the dosage for an adult human will bein the range 1 to 1000 milligrams, preferably 5-250 milligrams. Theactive compound can be administered with a conventional pharmaceuticalcarrier and may be administered orally, by injection or topically.

The preferred route of administration is oral administration. Foradministration by this route the compounds can be formulated into solidor liquid preparations, eg tablets, capsules, powders, solutions,suspensions and dispersions. Such preparations are well known in the artas are other oral dosage forms not listed here. In a preferredembodiment the compounds of the invention are tableted with conventionaltablet bases such as lactose, sucrose and corn starch together with abinder, a disintegration agent and a lubricant. These exipients are wellknown in the art. The binder may be for example corn starch or gelatin,the disintegrating agent may be potato starch or alginic acid and thelubricant may be magnesium stearate. Other components such as colouringagents and flavouring agents may be included.

Liquid forms for use in the invention include carriers such as water andethanol, with or without other agents such as a pharmaceuticallyacceptable surfactant or suspending agent.

The compounds of the invention may also be administered by injection ina physiologically acceptable diluent such as water or saline. Thediluent may comprise one or more of other ingredients such as ethanol,propylene glycol, an oil or a pharmaceutically acceptably surfactant.

Compounds of the invention may be applied to skin or mucous membranes.They may be present as ingredients in creams, preferably including apharmaceutically acceptable solvent to assist passage through the skinor mucous membranes. Suitable cream bases are well known to thoseskilled in the art.

The compounds of the invention may be administered by means of sustainedrelease systems for example they may be incorporated into a slowlydissolving tablet or capsule containing a solid or porous or matrix formfrom a natural or synthetic polymer.

BRIEF DESCRIPTION OF DRAWINGS

The following Figures are referred to in the Examples below.

FIG. 1 shows purine nucleoside phosphorylase activity with time at arange of concentrations of the product of Example 1 (Compound Ia).

FIG. 2 shows fitting of a purine nucleoside phosphorylase activityprogress curve to the kinetic model.

FIG. 3 shows K_(i) * determination by the curve fit method for CompoundIa inhibition of bovine purine nucleoside phosphorylase.

FIG. 4 shows a progress curve for bovine purine nucleoside phosphorylaseshowing slow-onset inhibition by Compound Ia.

EXAMPLES

The following examples further illustrate practice of the invention.Ratios of solvents are by volume.

Example 1--Preparation of(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitolExample 1.1

A solution of5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(Furneaux et al, Tetrahedron 53 (1997) 2915 and references therein) (2.0g) in pentane (40 ml) was stirred with N-chlorosuccinimide (1.2 g) for1h. The solids and solvent were removed and the residue was dissolved indry tetrahydrofuran (40 ml) and cooled to -78° C. A solution of lithiumtetramethylpiperidide (25 ml, 0.4 M in tetrahydrofuran) was added slowlydropwise. The resulting solution was then added via cannula to asolution of lithiated acetonitrile [prepared by the dropwise addition ofacetonitrile (2.08 ml, 40 mmol) to a solution of butyl lithium (29.8 ml,41.8 mmol) in dry tetrahydrofuran (50 ml) at -78° C., followed bystirring for 45 min and then addition of tetramethylpiperidine (0.67 ml,4 mmol)] at -78° C. The reaction mixture was stirred for 15 min thenquenched with water and partitioned between water and chloroform. Theorganic phase was dried and concentrated, and then chromatographyafforded(1S)-5-O-tert-butyldimethylsilyl-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(1) (0.83 g).

Example 1.2

A solution of the product from Example 1.1 (0.80 g) in dichloromethane(20 ml) containing di-tert-butyldicarbonate (0.59 g) was stirred at roomtemperature for 16 h. The solution was concentrated and thenchromatography afforded(1S)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(2) (0.89 g).

Example 1.3

To a solution of the product from Example 1.2 (0.88 g) inN,N-dimethylformamide (5 ml) was added tert-butoxybis(dimethylamine)methane (1.5 ml) and the solution was heated at 65-70°C. for 1 h. Toluene (20 ml) was added and the solution was washed (x3)with water, dried and concentrated to dryness. The residue was dissolvedin tetrahydrofuran/acetic acid/water (1:1:1 v/v/v, 40 ml) at roomtemperature. After 1.5 h chloroform (50 ml) was added and the mixturewas washed with water (x2), aqueous sodium bicarbonate, and then driedand evaporated to dryness. Chromatography of the residue gave(1S)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1-C-(1-cyano-2-hydroxyethenyl)-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(3) (0.68 g).

Example 1.4

Glycine hydrochloride ethyl ester (0.76 g) and sodium acetate (0.9 g)were added to a stirred solution of the product from Example 1.3 (0.51g) in methanol (10 ml). The mixture was stirred at room temperature for16 h and then concentrated to dryness. Chromatography of the residuegave the(1S)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1-C-[1-cyano-2-N-(ethoxycarbonylmethylamino)ethenyl]-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(4) (0.48) g as a diastereomeric mixture.

Example 1.5

A solution of the product from Example 1.4 (0.28 g) in drydichloromethane (12 ml) containing 1,8-diazabicyclo[5.4.0]undec-7-ene(1.5 ml) and benzyl chloroformate (0.74 ml) was heated under reflux for8 h, then cooled and washed with dilute aqueous HCl, aqueous sodiumbicarbonate, dried and concentrated. Chromatography of the residueafforded(1S)-1-C-[3-amino-1-N-benzyloxycarbonyl-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(5) (0.22 g).

Example 1.6

A solution of the product from Example 1.5 (0.22 g) in ethanol (10 ml)was stirred with 10% Pd/C (50 mg) in an atmosphere of hydrogen for 3 h.The solids and solvent were removed and the residue was dissolved inethanol (10 ml) containing formamidine acetate (0.40 g) and the solutionwas heated under reflux for 8 h. The solvent was removed andchromatography of the residue gave(1S)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1-C-[4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl]-1,4-imino-2,3-O-isopropylidene-D-ribitol(6) (156 mg).

Example 1.7

A solution of the product from Example 1.6 (66 mg) in trifluoroaceticacid (3 ml) was allowed to stand at room temperature overnight. Thesolution was concentrated and a solution of the residue in water waswashed (x2) with chloroform and then evaporated. The residue wasdissolved in methanol and treated with Amberlyst A21 base resin untilthe solution was pH-7. The solids and solvent were removed and theresidue was dissolved in water, treated with excess aqueous HCl and thenlyophilized. Trituration of the residue with ethanol gave(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol(7) hydrochloride salt as a white solid (25 mg). Recrystallised from 90%ethanol, the crystalline solid darkened but did not melt below 300° C.NMR (300 MHz, D₂ O with DCl, d ppm): ¹³ C (relative to internal acetoneat 33.2 ppm) 58.1 (C-1'), 61.4 (C-5'), 68.8 (C-4'), 73.3 (C-3'), 76.7(C-2'), 107.5 (q), 121.4 (q), 133.5 (C-2), 135.0 (q), 148.0 (C-6) and155.4 (q); ¹ H (relative to internal acetone at 2.20 ppm), 3.90(H-4'),3.96 (m, H-5',5"), 4.44 (dd, H-3', J_(2'),3' 5.4 Hz, J_(3'),4'3.2 Hz), 4.71 (dd, J_(1'),2' 9.0 Hz, H-2'), 5.00 (d, H-1'), 8.00 (s,H-6) and 9.04 (s, H-2). ##STR21##

Example 2--Preparation of(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitolExample 2.1

A solution of(1S)-1-C-[3-amino-1-N-benzyloxycarbonyl-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4dideoxy 1,4-imino-2,3-O-isopropylidene-D-ribitol (Example 1.5) (0.87 g)in ethanol was stirred with 10% Pd/C (100 mg) in an atmosphere ofhydrogen for 1.5 h. The solids and solvent were removed to give aresidue (0.61 g). To a solution of a portion of this residue (0.12 g) indichloromethane (10 ml) at 0° C. was added a solution of benzoylisothiocyanate in dichloromethane (31 mL in 1 ml). After 0.5 h thesolution was warmed to room temperature and1,8-diazabicyclo[5.4.0]undec-7-ene (80 mL) and methyl iodide (100 mL)were added. After another 0.5 h the reaction solution was applieddirectly to a silica gel column and elution afforded 0.16 g of(1S)-1-C-[3-(N-benzoyl-S-methylisothiocarbamoyl)amino-1-N-benzyloxycarbonyl-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol.

Example 2.2

A solution of this S-methylisothiocarbamoylamino derivative, (0.20 g) inmethanol saturated with ammonia was heated in a sealed tube at 95° C.for 16 h. The solvent was removed and chromatography of the residueafforded(1S)-1-C-[2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl]-N-tert-butoxycarbonyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol.

Example 2.3

A solution of this protected iminoribitol (64 mg) in trifluoroaceticacid was allowed to stand at room temperature for 16 h. The solvent wasremoved and a solution of the residue in aqueous methanol (1:1) wastreated with Amberlyst A21 base resin until the pH of the solution was˜7. The solids and solvent were removed and a solution of the residue inwater was treated with excess HCl and then concentrated to dryness.Trituration with ethanol gave(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitolhydrochloride salt (24 mg), which darkened at ca. 260° C. but did notmelt below 300° C. NMR (300 MHz, D₂ O with DCl, d ppm): ¹³ C (relativeto internal acetone at 33.1 ppm) 58.0 (C-1'), 61.4 (C-5'), 68.6 (C-4'),73.3 (C-3'), 76.3 (C-2'), 105.2 (q), 114.8 (q), 132.1 (C-6), 135.3 (q),153.4 (q) and 156.4 (q); ¹ H (relative to internal acetone at 2.20 ppm)3.87 (m, H-4'), 3.94 (m, H-5',5"), 4.40 (dd, J_(2'),3' 5.0 Hz,J_(3'),4', 3.2 Hz, H-3'), 4.65 (dd, J_(1'),2' 9.1 Hz, H-2'), 4.86 (d,H-1') and 7.71 (s, H-6).

Examples 3-24

The following compounds may be prepared according to methods disclosedin the general description:

3.(1R)-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-ezythro-pentitolmay be prepared from the product of Example 1 using Method H.

4.(1S)-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitolmay be prepared from the product of Example 1 using Method K.

5.(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-5-methylthio-D-ribitolmay be prepared from the product of Example 1 using Method K.

6.(1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitolmay be prepared from the product of Examples 1 or 2 using Method M.

7.(1R)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitolmay be prepared from the product of Example 6 using Method H.

8.(1S)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitolmay be prepared from the product of Example 6 using Method K.

9.(1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-5-methylthio-D-ribitolmay be prepared from the product of Example 6 using Method K.

10.(1R)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitolmay be prepared from the product of Example 2 by Method H.

11.(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitolmay be prepared from the product of Example 2 by Method K.

12.(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitolmay be prepared from the product of Example 2 using Method K.

13. (1S)-1,4-dideoxy-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-D-ribitol may be prepared by Methods D, E and F.

14.(1R)-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitolmay be prepared from the product of Example 13 using Method H.

15.(1S)-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitolmay be prepared from the product of Example 13 using Method K.

16. (1S)-1,4-dideoxy-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-5-methylthio-D-ribitol may be prepared from the product ofExample 13 using Method K.

17.(1S)-1,4-dideoxy-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-D-ribitolmay be prepared from the product of Example 13 using Method M.

18.(1R)-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitolmay be prepared from the product of Example 17 using Method H.

19.(1S)-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitolmay be prepared from the product of Example 17 using Method K.

20.(1S)-1,4-dideoxy-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-5-methylthio-D-ribitolmay be prepared from the product of Example 17 using Method K.

21.(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-D-ribitolmay be prepared using a variation of Method D in which the compound ofFormula XIa or XIb is replaced by a corresponding compound in which thehydrogen atom in position 5 is replaced by protected amino group.

22.(1R)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythropentitolmay be prepared from the product of Example 21 using Method H.

23.(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitolmay be prepared from the product of Example 21 using Method K.

24.(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitolmay be prepared from the product of Example 21 using Method K.

Example 25 Enzyme Assays

Enzyme assays were conducted to assess the effectiveness of the productsof Examples 1 and 2 (compounds Ia and Ib respectively) as inhibitors ofpurine nucleoside phosphorylase. The assays used human RBC and calfspleen purine nucleoside phosphorylase (ex Sigma, 90% pure) with inosineas substrate, in the presence of phosphate buffer, with detection ofreleased hypoxanthine using xanthine oxidase coupled reaction.

Inhibition of purine nucleoside phosphorylases

Materials. Inosine was obtained from Sigma. Xanthine oxidase (EC1.1.3.22, buttermilk), human erythrocyte (as a lyophilized powder) andbovine spleen (in 3.2 M ammonium sulfate) purine nucleosidephosphorylases (EC 2.4.2.1) were purchased from Sigma. Human purinenucleoside phosphorylases obtained as a powder was reconstituted in 100mM sodium phosphate buffer (pH 7.4) and rapidly frozen and stored at-80° C. Kinetic experiments were performed on a Uvikon 933 double beamultraviolet/visible spectrophotometer (Kontron Instruments, San Diego,Calif.).

Protein Concentrations. Protein concentrations for both isozymes weredetermined based on the quantative ultraviolet absorbance, using E_(1cm)1%=9.64 at 280 nm [Stoelkler et al, Biochemistry, 32 (1978) 278] and amonomer moleculer weight of 32,000 [Williams et al, Nucleic Acids Res.12 (1984) 5779].

Enzyme Assay. Enzymes were assayed spectrophotometrically using thecoupled xanthine oxidase method [Kalckar, J. Biol. Chem. 167 (1947) 429;Kim et al, J. Biol. Chem., 243 (1968) 1763]. Formation of uric acid wasmonitored at 293 nm. A 40 μM inosine solution gave an absorbance changeof 0.523 units at 293 m, upon complete conversion of inosine to uricacid and ribose 1-phosphate. Unless otherwise noted, the standard assayreaction contained: inosine (500 μM), potassium phosphate (50 mM, pH7.5); xanthine oxidase (0.06 units) and purine nucleoside phosphorylasein a final volume of 1.0 mL.

One-Third-the-Sites Inhibition. Reaction mixtures of 6.7 nM bovinepurine nucleoside phosphorylase and 40 μM inosine (3 times the K_(m)value) containing varying amounts of compound Ia were pre-incubated at30° C. for 1 hour. Reactions were initiated by addition of substrate andassayed at 30° C. The reaction containing 0.6 nM inhibitor(concentration ratio of [compound Ia]/[purine nucleosidephosphorylase]=0.09) showed 29% inhibition, that containing 1 nMinhibitor ([compound Ia]/[purine nucleoside phosphorylase]=0.15) showed44%, whereas the reaction containing 3 nM inhibitor ([compoundIa]/purine nucleoside phosphorylase]=0.44) had a rate decrease of 96%,and that containing 6 nM inhibitor ([compound Ia]/[purine nucleosidephosphorylate]=87%) showed 99% inhibition. These interactions are shownin FIG. 1.

Purine nucleoside phosphorylase is known to be a homotrimer with acatalytic site on each of the three protein subunits [Stoelkler et al,Biochemistry 32, (1978) 278]. When the concentration of enzyme subunitsis 6.7 nM, 50% inhibition of purine nucleoside phosphorylase occurs atapproximately 1.1 nM. This result demonstrates that compound Ia bindstightly and that binding of compound Ia to one site of the trimericenzyme leads to complete inhibition.

Activity Recovery from the Complex of Purine Nucleoside Phosphorylasewith Compound Ia. Purine nucleoside phosphorylase (6.7 μM) andsufficient compound Ia (3 μM) to inhibit 96% of purine nucleosidephosphorylase activity were incubated at 30° C. for 1 hour. An aliquotof this solution was diluted 1000-fold into a buffered solution of 500μM inosine containing xanthine oxidase (0.06 units). The production ofuric acid was monitored over time and the progres curve was fit to thekinetic model of FIG. 2.

Dilution of inhibited purine nucleoside phosphorylase into a largevolume of solution without inhibitor provided the tare of release ofcompound Ia from inhibited purine nucleoside phosphorylase. Underconditions of the experiment in FIG. 2, the time to achieve the newenzyme-inhibitor equilibrium is 5000 sec, an indication of a slow,tight-binding inhibitor [Morrison and Walsh, Advances Enzymol. 61 (1988)201]. The rate contants k₆ is an estimate of the apparent first-orderrate constant for dissociation of the complex under these experimentalconditions and is 2.9×10⁻⁴ sec⁻¹ in this example.

Inhibitory Mechanism. Slow, tight-binding inhibitors generally followthe kinetic mechanism [Morrison and Walsh, Advances Enzymol. 61 (1988)201]: ##STR22## where EI is a rapidly formed, initial collision complexof purine nucleoside phosphorylase (E) and compound Ia (I) that slowlyixomerizes to a tighter complex EI*. Product formation curves aredescribed by the following integrated rate equation 1:

    P=v.sub.s t+(v.sub.o -v.sub.s)(1-e.sup.-kt)/k              1

where P is the amount of product hypoxanthine (observed as uric acid inthe present assay system), t is time, v_(o) is the initial rate, v_(s)is the final steady-state rate and k is the overall (observed) rateconstant given by equation 2:

    k=k6+k5[(I/K.sub.i)/(1+(S/K.sub.m)+(I/K.sub.i))]           2

where K_(m) is the Michaelis complex for purine nucleosidephosphorylase, S is inosine concentration, I is the concentration ofcompound Ia and K_(i) is as described below. The rate of formation ofthe tightly bound complex is k5 and the rate of its dissociation is k6.KI, the inhibition constant for standard competitive inhibition (whichinfluences v_(o)) and K_(i) *, the overall inhibition constant (whichinfluences v_(s)), are defined as:

    K.sub.i =k4/k3

    K.sub.i *=K.sub.i [k.sub.6 /(k.sub.5 +k.sub.6)]

Determination of K_(i) *. K_(i) * was determined by measuring v_(s) forreactions at a range of inhibitor concentrations, plotting v_(s) vs [I]and fitting the curve to the competive inhibition equation 3:

    v.sub.s =V.sub.max S/[K.sub.m (1+I/K.sub.i *)+S]           3

where V_(max) is the uninhibited reaction rate for purine nucleosidephosphorylase, and the remaining terms are described above. The resultof this analysis indicates an overall effective inhibition constant(K_(i) *) of 2.5±0.2×10⁻¹¹ M (25±2pM) for compound Ia (FIG. 3).

Approximation of K_(i), k₅ and k₆. Calculation of K_(i) directly fromv_(o) and the competitive Inhibition equation (above) is difficult forcompound Ia because v_(o) changes very little as a function of I atinhibitor concentrations which cause complete inhibition following slowonset. This result establishes that the initial dissociation constantK_(i) is much greater than the equilibrium dissociation constant K_(i)*.

Approximations of k₅ and K_(i) were calculated from k (values obtainedfrom curve fits of equation 1, FIG. 4) by using equation 2. Using theknowledge that k₆ <<k₅ [(I/K_(i))/(1+(A/K_(m))+(I/K_(i))], equation 2can be rearranged so that a double reciprocal plot of 1/k vs 1/[I] givesa straight line with y intercept=1/k₅ and x intercept of -(1/k₅)/[K_(i)/k₅)*(A/K_(m)))]. Substitution of these values into equation 2 give anapproximation for k₆. FIG. 4 demonstrates the slow-onset, tight-bindinginhibition which occurs when a small concentration of enzyme (0.8 nM)competes for 200 nM compound Ia in the presence of 500 μM inosine. Underthese conditions the apparent first order rate constant for onset ofinhibition in FIG. 4 was 26×10⁻⁴ sec⁻¹.

The result of FIG. 4 demonstrates that even at inosine concentrationsover 100 times that present in human serum or tissues, compound Ia cangive 99% inhibition of the enzyme after several minutes of slow-onsetinhibition. Based on analyses of experiments of the type shown in FIGS.1-4, the experimentally estimated dissociation constants and rates forthe bovine purine nucleoside phosphorylase with compound Ia are:

    K.sub.m =15 μM

    K.sub.i =19±4 nM

    K.sub.i *=25±2 pM

    k.sub.5 =1.4±0.2×10.sup.-2 sec.sup.-1

    k.sub.6 =1.8±0.5×10.sup.-5 sec.sup.-1

Inhibition of Human Purine Nucleoside Phosphorylase. Studies similar tothose described above for the interaction of bovine purine nucleosidephosphorylase were conducted with purine nucleoside phosphorylase (PNP)from human erythrocytes. The values for the overall inhibition constant,K_(i) *, for the interaction of human and bovine PNP with compound Iaare:

    ______________________________________                                        enzyme             K.sub.i *, compound Ia                                     ______________________________________                                        human PNP          100 pM                                                       bovine PNP  25 pM, 17 pM.sup.a                                              ______________________________________                                         .sup.a The results of two different experimental analyses.               

The compound Ib is a more efficient inhibitor for the human enzyme thancompound Ia, but compound Ia is slightly more efficient at inhibitingthe bovine enzyme.

Summary of Compounds Ia and Ib as Inhibitors of Purine NucleosidePhosphorylases.

Inhibitors usually function by binding at every catalytic site to causefunctional inhibition in living organisms. The one-third-the-sitesinhibition and the slow-onset tight-binding inhibition described aboveindicate that compounds Ia and Ib are very potent inhibitors of purinenucleoside phosphorylases able to function in the presence of a largeexcess of substrate.

The methods for the determination of the kinetic constants are given indetail in Merkler, D. J., Brenowitz, M, and Schramm, V. L. Biochemistry29 (1990) 8358-8364.

Inhibition of Protozan Nucleoside Hydrolases by Compounds Ia and Ib.

Protozan parasites use the hydrolysis of purine nucleosides such asinosine to provide purine bases such as hypoxanthine to provideessential precursors for RNA and DNA synthesis. Protozoan parasites arepurine auxotrophs. Using inhibition methods similar to those describeabove, a nucleoside hydrolase from Crithidia fasciculata [Parkin, et al,J Biol, Chem. 266 (1991) 20658] and a nucleoside hydrolase fromTrypanosoma brucei brucei [Parkin, J. Biol. Chem. (1996) 21713] weretested for inhibition by compounds Ia and Ib. The results are summarisedbelow.

    ______________________________________                                                      K.sub.i values                                                  enzyme          compound Ia                                                                             compound Ib.sup.a                                   ______________________________________                                        nucleoside hydrolase                                                                          29 nM     127 nM                                                C. fasciculata                                                                nucleoside hydrolase 23 nM -200 nM.sup.a                                      T. brucei brucei                                                            ______________________________________                                         .sup.a The results give complex kinetic plots and this values is an           estimate.                                                                

The inhibitors bind in direct competition with substrate, therefore theinhibition constants are direct competitive inhibition values. Thecompounds provide sufficient inhibition to protozoan parasites toinhibit the purine nucleoside hydrolases at readily accessiblepharmacological doses.

The methods and materials used are as described in published PCTinternational application WO 97/31008 using p-nitrophenyl riboside assubstrate.

Example 26--Tablet

4 grams of the product of Example 1 is mixed with 96 grams of lactoseand 96 grams of starch. After screening and mixing with 2 grams ofmagnesium stearate, the mixture is compressed to give 250 milligramtablets.

Example 27--Gelatin Capsule

Ten grams of the product of Example 1 is finely ground and mixed with 5grams of talc and 85 grams of finely ground lactose. The powder isfilled into hard gelatin capsules.

Aspects of the invention have been described by way of example only andit should be appreciated that modifications and additions thereto may bemade without departing from the scope of the invention.

What is claimed is:
 1. A compound having the formula: ##STR23## whereinA is CH or N; B is chosen from OH, NH₂, NHR, H or halogen; D is chosenfrom OH, NH₂, NHR, H, halogen or SCH₃ ; R is an optionally substitutedalkyl, aralkyl or aryl group; and X and Y are independently selectedfrom H, OH or halogen except that when one of X and Y is hydroxy orhalogen, the other is hydrogen; and Z is OH or, when X is hydroxy, Z isselected from hydrogen, halogen, hydroxy, SQ or OQ where Q is anoptionally substituted alkyl, aralkyl or aryl group; or a tautomerthereof; or a pharmaceutically acceptable salt thereof; or an esterthereof; or a prodrug thereof.
 2. The compound of claim 1, wherein oneof B and/or D is NHR, and R is C₁ -C₄ alkyl.
 3. The compound of claim 1,wherein either D is H, or B is OH, or both.
 4. The compound of claim 1,wherein B is OH, D is H, OH or NH₂, X is OH or H, Y is H.
 5. Thecompound of claim 4, wherein Z is OH, H or methylthio.
 6. The compoundof claim 5, wherein Z is OH.
 7. The compound of claim 1 selectedfrom(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol,(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitol,(1R)-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol,(1S)-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol,(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-5-methylthio-D-ribitol,(1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol,(1R)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythropentitol,(1S)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol,(1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-5-methylthio-D-ribitol,(1R)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol,(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol,(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol,(1S)-1,4-dideoxy-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-D-ribitol,(1R)-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythropentitol,(1S)-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol,(1S)-1,4-dideoxy-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-5-methylthio-D-ribitol,(1S)-1,4-dideoxy-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-D-ribitol,(1R)-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol,(1S)-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol,(1S)-1,4-dideoxy-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-5-methylthio-D-ribitol,(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-D-ribitol,(1R)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol,(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol,and(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol,ora tautomer thereof; or a pharmaceutically acceptable salt thereof. 8.The compound of claim 1 which is(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol,or tautomer thereof, or a pharmaceutically acceptable salt thereof. 9.The compound of claim 1 which is (1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitol, or tautomer thereof,or a pharmaceutically acceptable salt thereof.
 10. A pharmaceuticalcomposition for the suppression of T-cell function comprising an amountof a compound of claim 1 effective for inhibiting purine nucleosidephosphorylase, and a pharmaceutically acceptable carrier or diluent. 11.A pharmaceutical composition for the suppression of T-cell functioncomprising an amount of a compound of claim 2 effective for inhibitingpurine nucleoside phosphorylase, and a pharmaceutically acceptablecarrier or diluent.
 12. A pharmaceutical composition for the suppressionof T-cell function comprising an amount of a compound of claim 3effective for inhibiting purine nucleoside phosphorylase, and apharmaceutically acceptable carrier or diluent.
 13. A pharmaceuticalcomposition for the suppression of T-cell function comprising an amountof a compound of claim 4 effective for inhibiting purine nucleosidephosphorylase, and a pharmaceutically acceptable carrier or diluent. 14.A pharmaceutical composition for the suppression of T-cell functioncomprising an amount of a compound of claim 5 effective for inhibitingpurine nucleoside phosphorylase, and a pharmaceutically acceptablecarrier or diluent.
 15. A pharmaceutical composition for the suppressionof T-cell function comprising an amount of a compound of claim 6effective for inhibiting purine nucleoside phosphorylase, and apharmaceutically acceptable carrier or diluent.
 16. A pharmaceuticalcomposition for the suppression of T-cell function comprising an amountof a compound of claim 7 effective for inhibiting purine nucleosidephosphorylase, and a pharmaceutically acceptable carrier or diluent. 17.A pharmaceutical composition for the suppression of T-cell functioncomprising an amount of a compound of claim 8 effective for inhibitingpurine nucleoside phosphorylase, and a pharmaceutically acceptablecarrier or diluent.
 18. A pharmaceutical composition for the suppressionof T-cell function comprising an amount of a compound of claim 9effective for inhibiting purine nucleoside phosphorylase, and apharmaceutically acceptable carrier or diluent.
 19. A pharmaceuticalcomposition for treatment and/or prophylaxis of a protozoan infectioncomprising an amount of a compound of claim 1 effective for inhibitingat least one parasite purine nucleoside hydrolase or purine nucleosidephosphorylase and a pharmaceutically acceptable carrier diluent.
 20. Amethod for decreasing T-cell function in a mammal comprisingadministering to the mammal a compound of claim 1, whereby said compoundinhibits purine nucleoside phosphorylase.
 21. A method for treatmentand/or prophylaxis of an infection caused by protozoan parasitecomprising administering to a subject an amount of a compound of claim 1effective to inhibit at least purine nucleoside hydrolase or purinenucleoside phosphorylase.
 22. A method for killing parasites comprisingadministering the parasite an amount of a compound of claim 1 effectivefor inhibiting at least one purine nucleoside hydrolase or purinenucleoside phosphorylase.